In many fields of science, purified compounds are required for testing and analysis protocols. Purification of a compound involves separating out a desired component or components from a mixture that contains additional components or impurities. Chromatography is a method of fractionating a mixture to separate components of the mixture. In liquid chromatography, a sample containing a number of components to be separated is injected into a fluid stream, and directed through a chromatographic column. The column is so designed that it separates the mixture, by differential retention on the column, into its component species. The different species then emerge from the column as distinct bands, separated in time.
A typical high performance liquid chromatography system (HPLC system) is comprised of a pump for delivering fluids (the “mobile phase”) at a controlled flow rate and composition, an injector to introduce a sample solution into the flowing mobile phase, a tubular column encasement containing a packing material or sorbent (the “stationary phase”), and a detector to register the presence and amount of the sample components in the mobile phase. When the mobile phase is passed through the stationary phase, each component of the sample will emerge from the column at a different time because different components in the sample will have different affinities for the packing material. The presence of a particular component in the mobile phase exiting the column can be detected by measuring changes in physical or chemical properties of the eluent. By plotting the detector's signal over time, response “peaks” corresponding to the presence of each of the components of the sample can be observed and recorded.
Preparative HPLC is a convenient and easy way of isolating and purifying a quantity of a compound for further studies. Preparative HPLC is not limited by scale. Depending on the specific application, Preparative separations can involve very large columns and sample sizes, resulting in multigram yields, or may be performed using very small columns, resulting in microgram yields. Thus, a common distinction between Preparative and Analytical HPLC is that in Preparative HPLC, the sample is collected after purification, whereas in Analytical HPLC, the sample components are simply detected and quantified.
Typical target requirements for a Preparative separation include high recovery of the sample compound at a purity exceeding 90%, and a rapid, efficient routine. A single instrument is required to isolate and purify between fifty and one hundred samples per day. Therefore, it is highly desirable to purify and separate the largest possible quantity of sample with each run, thereby reducing labor, space, operating expense, run time and associated instrumentation costs.
The combination of Preparative HPLC with a mass spectrometer permits a large number of samples to be processed automatically. An automated separation scheme is guided by mass spectrometer detection. The mass spectrometer is set up to detect the expected molecular weight of the target sample and direct the collection of the purified component that contains this molecular weight.
Other approaches to reduce run time in a Preparative HPLC system include providing rapid gradients during gradient elution, and employing a shorter column with smaller particles in the stationary phase. However, these techniques often compromise the results of the analysis, leading to loss of resolution and a deterioration of peak shape, indicating decreased purity of the separated sample.
In Preparative chromatography, it is also desirable to maximize the quantity of sample to be separated per volume of packing material in the column. Smaller volume columns contain less packing material, which often will have a significant impact on the cost of the column. However, the resolution between response peaks in a chromatographic analysis or “run” depends, in part, on the loading capacity of the column. Chromatography results are limited by the loading capacity of the column, defined as a threshold for the maximum volume and/or mass of sample that may be loaded onto the column without compromising results.
The loading capacity for a column can be exceeded in two ways: volume overload; and mass overload. Volume overload can be defined as the volume of injected sample solution where loss of resolution occurs. Mass overload can be defined as the mass of solute in the sample solution above which loss of resolution occurs.
Loading capacity of a column can be measured by injecting a progressively larger amount of sample. Often times, compounds will elute with multiple peaks having different retention times. A load that exceeds the column capacity is characterized by a deterioration of peak shape and loss of resolution in the resulting separation.
The column can be any chromatographic column, either of conventional or cartridge design. The column can also be composed of two or more columns that are interconnected in some way. An example of such an arrangement would be the use of an easily replaceable guard column connected in series upstream from another column, thereby protecting the main column from premature failure due to fouling.
Neue et al. (Advances in Chromatography, 41: 93-136 (2001), the disclosure of which is hereby incorporated herein by reference) describe techniques for optimizing a reversed-phase gradient separation by varying the column length, particle sizes and running conditions for the separation. The article further describes optimized sample loading schemes for providing simplified and automatable preparative gradient elution.
It is desirable to enhance the loading capacity of a column, thereby allowing for the purification and isolation of a larger quantity of a purified sample per chromatographic run. An increased loading capacity for a chromatography column also implies less run time required, and lower cost associated with the isolation of a fixed quantity of sample.